The cellular hemoglobin of red blood cells is an important parameter for clinical diagnosis. On hematology analyzers, Mean corpuscular hemoglobin (MCH) of a blood sample is derived from the red blood cell count (RBC) and total hemoglobin (Hgb) of the blood sample. The latter is obtained by a spectrophotometric measurement of hemoglobin concentration of a lysed blood sample. MCH is an average measurement of all red blood cells, it does not represent hemoglobin content of individual red blood cells.
In contrast, the measurement of cellular hemoglobin in a flow cytometer is a cell-by-cell measurement, which provides diagnostic information that is not available through MCH. Campbell et al. Cytometry 35, pp 242-248 (1999) have performed flow cytometric analysis of hemoglobin in individual red blood cells using a fluorescent anti-hemoglobin A antibody. Burshteyn et al. (U.S. Patent Application Publication No. 2004/0214243) have performed flow cytometric analysis of hemoglobin using an anti-pan hemoglobin antibody. Since red blood cells have a very high concentration of hemoglobin, in order to measure the total cellular hemoglobin using antibodies, a large amount of fluorescent antibodies are required. Furthermore, there are potential artifacts due to steric hindrance of antibody binding or extinction of fluorescence due to the high density of hemoglobin in the cell. Therefore, it is desirable to have a method that enables the measurement of total cellular hemoglobin without relying on the use of antibodies.
On the other hand, cell-by-cell hemoglobin measurement has been developed in hematology analysis using multi-angle light scatter measurements. To facilitate the light scatter measurement, typically a blood sample is treated with an isotonic, neutral sphering reagent to sphere the red blood cells prior to the measurement. When untreated red blood cells are measured by light scatter measurements, they produce heterogeneous scatter results, because the concave shaped cells can have various different orientations as they pass through the flow cell. Upon mixing with a sphering reagent, the red blood cells are sphered, which can produce homogeneous scatter results. However, the light scatter measurement for determining cellular hemoglobin is complex, typically multiple angles of light scatter signals are utilized. For example, Bayer's hematology analyzers provide a cell-by-cell measurement of red blood cells using a complex light scatter measurement based on Mei theory. Although all commercial available flow cytometers are equipped with forward and side scatter measurement devices, the forward and side scatter signals of sphered red blood cells do not correlate to the cellular hemoglobin, and hence have not been used for quantitative measurement of cellular hemoglobin. Furthermore, the red blood cells treated by sphering reagent are not permeable to large intracellular markers such as antibodies, therefore, not suitable for measurement of hemoglobin variants utilizing antibodies.
Identifying and/or quantifying variants and aberrant forms of hemoglobin are important for clinical diagnosis of various diseases, for example, sickle cell disease, thalassemia and diabetics. The measurement of hemoglobin A1C has been one of most frequently used hemoglobin variant measurement, and is an important clinical measure for diabetic patients.
It is known that about 90% of total hemoglobin is nonglycosylated. The major fraction of nonglycosylated hemoglobin is nonglycosylated HbA, referred to as HbA0. Glycated hemoglobin refers to a series of minor hemoglobin components that are formed via the attachment of various sugars to the hemoglobin molecule. The human erythrocyte is freely permeable to glucose. Within each erythrocyte, glycated hemoglobin is formed at a rate that is directly proportional to the ambient glucose concentration. The reaction of glucose with hemoglobin is nonenzymatic, irreversible and slow, so that only a fraction of the total hemoglobin is glycated during the life span of an erythrocyte (120 days). As a result, the measurement of glycated hemoglobin provides a weighted “moving” average of blood glucose levels that can be used to monitor long-term blood glucose levels, providing an accurate index of the mean blood glucose concentration over the preceding 2 to 3 months. The most important clinical application of this is in the assessment of glycemic control in a diabetic patient.
Hemoglobin A1C (HbA1C) is one specific type of glycated hemoglobin and is the most important hemoglobin species with respect to diabetes. HbA1C is approximately 3 to 6% of the total hemoglobin in nondiabetics, and 20% or greater in diabetes that is poorly controlled (Goldstein, et al., Clin. Chem. 32: B64-B70, 1986). The determination of the concentration of HbA1C is useful in diagnosing and monitoring diabetes mellitus.
Based on the above, it is therefore desirable to have a method that enables measurements of total hemoglobin content and specific hemoglobin variant in individual red blood cells at the same time. Moreover, it is further desirable to have a method that enables such a cell-by-cell measurement for different red blood cell subpopulations, such as in both mature red blood cells, and reticulocytes, at the same time.